PROJECT ABSTRACT Stimulant drugs induce wide-spread epigenetic perturbations in the brain, but their effects on the epigenetic regulation of the latent HIV provirus and responses to latency-reversing therapies targeting this regulation are unknown. The central hypothesis of this application is that histone methylation by the SET and MYND domain- containing enzyme Smyd2 (also called KMT3C) is a robust latency-inducing mechanism and a new target for latency-reversing therapy in HIV+ cocaine users. This hypothesis was formulated on the basis of preliminary results generated by the applicant identifying Smyd2, among 45 cellular lysine methyltransferases, as a top enzyme that suppresses HIV transcription in latent CD4+ T cells. It is also based on published work by others showing that expression of G9a, another methyltransferase linked to HIV latency, is decreased in the brain after chronic cocaine exposure, while expression of Smyd2 is unchanged. The central hypothesis will be tested in three specific aims: 1) Define how Smyd2 functions as transcriptional repressor in HIV latency. The working hypothesis is that Smyd2, by associating with the latent-HIV promoter in vivo, induces durable transcriptional repression despite cocaine exposure. The applicant will test this hypothesis with CRISPR/Cas9 gene?editing technology and by examining the mode of Smyd2 recruitment with and without cocaine exposure. She will also test latency reversal in response to small-molecule Smyd2 inhibitors in CD4+ T cells isolated from HIV+ cocaine users. 2) Determine the lysine methylation mark(s) set by Smyd2 at the latent HIV promoter. The working hypothesis is that monomethylation of lysine 20 at histone H4 (H4K20me1) is the main mark set by Smyd2 at the latent viral promoter during cocaine exposure. The applicant will test this hypothesis by determining the methyl marks introduced by Smyd2 using mass spectrometry and by performing chromatin immunoprecipitations (ChIP) for Smyd2 histone?methyl marks in latent cells with and without cocaine treatment. 3) Identify the mechanism underlying how H4K20me1 induces HIV latency. The working hypothesis is that H4K20me1 at the latent provirus is bound by the MBT-containing protein L3MBTL1, which induces chromatin compaction and, thus, durable transcriptional repression and latency with cocaine exposure. The applicant will test this hypothesis by performing ChIP and histone accessibility assays (ATAC-Seq) to measure L3MBTL1 recruitment and chromatin compaction, respectively, in latent cells with and without cocaine exposure. Successful completion of this proposal will significantly enhance the understanding of epigenomic regulatory mechanisms in HIV/AIDS infection in combination with substance abuse in alignment with this RFA. The proposed research is innovative because it represents a new and substantive departure from the status quo by shifting the focus in HIV latency research to monomethylation of histone H4, recruitment of L3MBTL1, and induction of histone compaction. Based on this work, the applicant expects to make important advances in therapies for HIV+ drug users and in improving the understanding of the epigenetics of drug use.